Blood-brain Barrier Homeostasis Research Articles

Casein Kinase 2 Inhibitor, CX-4945, Induces Apoptosis and Restores Blood-Brain Barrier Homeostasis in In Vitro and In Vivo Models of Glioblastoma.

Background: In oncology, casein kinase 2 (CK2), a serine/threonine kinase, has a dual action, regulating cellular processes and acting as an oncogenic promoter. Methods: This study examined the effect of CX-4945, a selective CK2 inhibitor, in a human U-87 glioblastoma (GBM) cell line, treated with CX-4945 (5, 10, and 15 μM) for 24 h. Similarly, the hCMEC/D3 cell line was used to mimic the blood-brain barrier (BBB), examining the ability of CX-4945 to restore BBB homeostasis, after stimulation with lipopolysaccharide (LPS) and then treated with CX-4945 (5, 10, and 15 μM). Results: We reported that CX-4945 reduced the proliferative activity and modulated the main pathways involved in tumor progression including apoptosis. Furthermore, in confirmation of the in vitro study, performing a xenograft model, we demonstrated that CX-4945 exerted promising antiproliferative effects, also restoring the tight junctions' expression. Conclusions: These new insights into the molecular signaling of CK2 in GBM and BBB demonstrate that CX-4945 could be a promising approach for future GBM therapy, not only in the tumor microenvironment but also at the BBB level.

Type-1-to-type-2 transition of brain microvascular pericytes induced by cytokines and disease-associated proteins: Role in neuroinflammation and blood-brain barrier disruption.

While the concept of pericyte heterogeneity in the brain microvasculature is becoming more widely accepted, little is known about how they arise, or their functional contributions to the blood-brain barrier (BBB). We therefore set out to examine the distribution of subtypes of pericytes at the BBB and sought to elucidate some of their functional characteristics by examining their unique mRNA expression patterns. We demonstrate that type-1 pericytes (PC1) that are associated with young healthy brains and BBB homeostasis, can transition into type-2 pericytes (PC2) that are associated with disease and BBB breakdown, both in vitro and in vivo, in the presence of both endogenous and disease associated ligands. We identified PC1 and PC2 in single-cell RNA-sequencing from vascular enriched mouse brain and identified transcriptional differences between PC1 and PC2. PC2 showed increased expression of genes associated with phagocytosis and peripheral immune cell infiltration. On the contrary, PC1 displayed increased expression of genes involved in hedgehog signaling, which is known to promote tight junction formation at the BBB. Our data support the PC1-to-PC2 transition as an origin of PC diversity and suggest a functional role for PC1 in maintaining BBB homeostasis and PC2 in responding to pathological conditions.

Progress of researches on the mechanisms of acupuncture intervention underlying improvement of ischemic stroke by regulating microRNAs.

MicroRNAs play an important role in the occurrence and development of ischemic stroke (IS). A lot of researches have shown that acupuncture intervention can improve IS-induced neural dysfunction by regulating miRNA. In the present paper, we summarized the current progress of researches on the mechanisms of acupuncture underlying improvement of IS via regulation of miRNA from 1) promoting angiogenesis and increasing cerebral blood flow, 2) inhibiting inflammatory response, 3) maintaining blood-brain barrier homeostasis and relieving brain edema, 4) regulating programmed cell death, 5) promoting neuron regeneration, and 6) improving synaptic plasticity. These miRNA -related mechanisms may provide a reference for the follow-up research .

Endothelial lincRNA-p21 alleviates cerebral ischemia/reperfusion injury by maintaining blood-brain barrier integrity

Blood-brain barrier (BBB) disruption is increasingly recognized as an early contributor to the pathophysiology of cerebral ischemia/reperfusion (I/R) injury, and is also a key event in triggering secondary damage to the central nervous system. Recently, long non-coding RNA (lncRNA) have been found to be associated with ischemic stroke. However, the roles of lncRNA in BBB homeostasis remain largely unknown. Here, we report that long intergenic non-coding RNA-p21 (lincRNA-p21) was the most significantly down-regulated lncRNA in human brain microvascular endothelial cells (HBMECs) after oxygen and glucose deprivation/reoxygenation (OGD/R) treatment among candidate lncRNA, which were both sensitive to hypoxia and involved in atherosclerosis. Exogenous brain-endothelium-specific overexpression of lincRNA-p21 could alleviate BBB disruption, diminish infarction volume and attenuate motor function deficits in middle cerebral artery occlusion/reperfusion (MCAO/R) mice. Further results showed that lincRNA-p21 was critical to maintain BBB integrity by inhibiting the degradation of junction proteins under MCAO/R and OGD/R conditions. Specifically, lincRNA-p21 could inhibit autophagy-dependent degradation of occludin by activating PI3K/AKT/mTOR signaling pathway. Besides, lincRNA-p21 could inhibit VE-cadherin degradation by binding with miR-101-3p. Together, we identify that lincRNA-p21 is critical for BBB integrity maintenance, and endothelial lincRNA-p21 overexpression could alleviate cerebral I/R injury in mice, pointing to a potential strategy to treat cerebral I/R injury.

Alterations of the blood-brain barrier during aging.

The blood-brain barrier (BBB) is a complex and dynamic interface that regulates the exchange of molecules and cells between the blood and the central nervous system. It undergoes structural and functional changes during aging, which may compromise its integrity and contribute to the pathogenesis of neurodegenerative diseases. In recent years, advances in microscopy and high-throughput bioinformatics have allowed a more in-depth investigation of the aging mechanisms of BBB. This review summarizes age-related alterations of the BBB structure and function from six perspectives: endothelial cells, astrocytes, pericytes, basement membrane, microglia and perivascular macrophages, and fibroblasts, ranging from the molecular level to the human multi-system level. These basic components are essential for the proper functioning of the BBB. Recent imaging methods of BBB were also reviewed. Elucidation of age-associated BBB changes may offer insights into BBB homeostasis and may provide effective therapeutic strategies to protect it during aging.

The role of platelets in the blood-brain barrier during brain pathology.

Platelets play critical roles in maintaining hemostasis. The blood brain barrier (BBB), a significant physical and metabolic barrier, helps maintain physiological stability by limiting transportations between the blood and neural tissues. When the brain undergoes inflammation, tumor, trauma, or bleeding, the platelet responses to help with maintaining BBB homeostasis. In the traditional point of view, activated platelets aggregate to form thrombi which cover the gaps of the blood vessels to protect BBB. However, increasing evidences indicate that platelets may harm BBB by enhancing vascular permeability. Hereby, we reviewed recently published articles with a special focus on the platelet-mediated damage of BBB. Factors released by platelets can induce BBB permeability, which involve platelet-activating factors (PAF), P-selectin, ADP, platelet-derived growth factors (PDGF) superfamily proteins, especially PDGF-AA and PDGF-CC, etc. Platelets can also secrete Amyloid-β (Aβ), which triggers neuroinflammation and downregulates the expression of tight junction molecules such as claudin-5 to damage BBB. Additionally, platelets can form aggregates with neutrophils to release reactive oxygen species (ROS), which can destroy the DNA, proteins, and lipids of endothelial cells (ECs). Moreover, platelets participate in neuroinflammation to affect BBB. Conversely, some of the platelet released factors such as PDGF-BB, protects BBB. In summary, platelets play dual roles in BBB integrity and the related mechanisms are reviewed.

MFSD2A frameshift variant in Kerry Hill sheep with microcephaly.

Microcephaly is a rare neurodevelopmental disorder characterized by reduced skull circumference and brain volume that occurs sporadically in farm animals. We investigated an early-onset neurodegenerative disorder observed in seven lambs of purebred Kerry Hill sheep. Clinical signs included inability to stand or severe ataxia, convulsions, and early death. Diagnostic imaging and brain necropsy confirmed microcephaly. The pedigree of the lambs suggested monogenic autosomal recessive inheritance. We sequenced the genome of one affected lamb, and comparison with 115 control genomes revealed a single private protein-changing variant. This frameshift variant, MFSD2A: c.285dupA, p.(Asp96fs*9), represents a 1-bp duplication predicted to truncate 80% of the open reading frame. MFSD2A is a transmembrane protein that is essential for maintaining blood-brain barrier homeostasis and plays a key role in regulating brain lipogenesis. Human MFSD2A pathogenic variants are associated with a neurodevelopmental disorder with progressive microcephaly, spasticity, and brain imaging abnormalities (NEDMISBA, OMIM 616486). Here we present evidence for the occurrence of a recessively inherited form of microcephaly in sheep due to a loss-of-function variant in MFSD2A (OMIA 002371-9940). To the best of our knowledge, this is the first report of a spontaneous MFSD2A variant in domestic animals.

Endothelial depletion of Atg7 triggers astrocyte-microvascular disassociation at blood-brain barrier.

Microvascular basement membrane (BM) plays a pivotal role in the interactions of astrocyte with endothelium to maintain the blood-brain barrier (BBB) homeostasis; however, the significance and precise regulation of the endothelial cell-derived BM component in the BBB remain incompletely understood. Here, we report that conditional knockout of Atg7 in endothelial cells (Atg7-ECKO) leads to astrocyte-microvascular disassociation in the brain. Our results reveal astrocytic endfeet detachment from microvessels and BBB leakage in Atg7-ECKO mice. Furthermore, we find that the absence of endothelial Atg7 downregulates the expression of fibronectin, a major BM component of the BBB, causing significantly reduced coverage of astrocytes along cerebral microvessels. We reveal Atg7 triggers the expression of endothelial fibronectin via regulating PKA activity to affect the phosphorylation of cAMP-responsive element-binding protein. These results suggest that Atg7-regulated endothelial fibronectin production is required for astrocytes adhesion to microvascular wall for maintaining the BBB homeostasis. Thus, endothelial Atg7 plays an essential role in astrocyte-endothelium interactions to maintain the BBB integrity.

Highlighting In Vitro the Role of Brain-like Endothelial Cells on the Maturation and Metabolism of Brain Pericytes by SWATH Proteomics

Within the neurovascular unit, brain pericytes (BPs) are of major importance for the induction and maintenance of the properties of the blood-brain barrier (BBB) carried by the brain microvessel endothelial cells (ECs). Throughout barriergenesis, ECs take advantage of soluble elements or contact with BPs to maintain BBB integrity and the regulation of their cellular homeostasis. However, very few studies have focused on the role of ECs in the maturation of BPs. The aim of this study is to shed light on the proteome of BPs solocultured (hBP-solo) or cocultured with ECs (hBP-coc) to model the human BBB in a non-contact manner. We first generated protein libraries for each condition and identified 2233 proteins in hBP-solo versus 2492 in hBP-coc and 2035 common proteins. We performed a quantification of the enriched proteins in each condition by sequential window acquisition of all theoretical mass spectra (SWATH) analysis. We found 51 proteins enriched in hBP-solo related to cell proliferation, contractility, adhesion and extracellular matrix element production, a protein pattern related to an immature cell. In contrast, 90 proteins are enriched in hBP-coc associated with a reduction in contractile activities as observed in vivo in 'mature' BPs, and a significant gain in different metabolic functions, particularly related to mitochondrial activities and sterol metabolism. This study highlights that BPs take advantage of ECs during barriergenesis to make a metabolic switch in favor of BBB homeostasis in vitro.

Understanding drug nanocarrier and blood-brain barrier interaction based on a microfluidic microphysiological model.

As many nanoparticles (NPs) have been exploited as drug carriers to overcome the resistance of the blood-brain barrier (BBB), reliable in vitro BBB models are urgently needed to help researchers to comprehensively understand drug nanocarrier-BBB interaction during penetration, which can prompt pre-clinical nanodrug exploitation. Herein, we developed a microfluidic microphysiological model, allowing the analysis of BBB homeostasis and NP penetration. We found that the BBB penetrability of gold nanoparticles (AuNPs) was size- and modification-dependent, which might be caused by a distinct transendocytosis pathway. Notably, transferrin-modified 13 nm AuNPs held the strongest BBB penetrability and induced the slightest BBB dysfunction, while bare 80 nm and 120 nm AuNPs showed opposite results. Moreover, further analysis of the protein corona showed that PEGylation reduced the protein absorption, and some proteins facilitated the BBB penetration of NPs. The developed microphysiological model provides a powerful tool for understanding the drug nanocarrier-BBB interaction, which is vital for exploiting high-efficiency and biocompatible nanodrugs.

Genome Editing with AAV-BR1-CRISPR in Postnatal Mouse Brain Endothelial Cells.

Brain endothelial cells (ECs) are an important component of the blood-brain barrier (BBB) and play key roles in restricting entrance of possible toxic components and pathogens into the brain. However, identifying endothelial genes that regulate BBB homeostasis remains a time-consuming process. Although somatic genome editing has emerged as a powerful tool for discovery of essential genes regulating tissue homeostasis, its application in brain ECs is yet to be demonstrated in vivo. Here, we used an adeno-associated virus targeting brain endothelium (AAV-BR1) combined with the CRISPR/Cas9 system (AAV-BR1-CRISPR) to specifically knock out genes of interest in brain ECs of adult mice. We first generated a mouse model expressing Cas9 in ECs (Tie2Cas9). We selected endothelial β-catenin (Ctnnb1) gene, which is essential for maintaining adult BBB integrity, as the target gene. After intravenous injection of AAV-BR1-sgCtnnb1-tdTomato in 4-week-old Tie2Cas9 transgenic mice resulted in mutation of 36.1% of the Ctnnb1 alleles, thereby leading to a dramatic decrease in the level of CTNNB1 in brain ECs. Consequently, Ctnnb1 gene editing in brain ECs resulted in BBB breakdown. Taken together, these results demonstrate that the AAV-BR1-CRISPR system is a useful tool for rapid identification of endothelial genes that regulate BBB integrity in vivo.

Understanding the Heterogeneity of Human Pericyte Subsets in Blood–Brain Barrier Homeostasis and Neurological Diseases

Pericytes are increasingly recognized as being important in the control of blood–brain barrier permeability and vascular flow. Research on this important cell type has been hindered by widespread confusion regarding the phenotypic identity and nomenclature of pericytes and other perivascular cell types. In addition, pericyte heterogeneity and mouse–human species differences have contributed to confusion. Herein we summarize our present knowledge on the identification of pericytes and pericyte subsets in humans, primarily focusing on recent findings in humans and nonhuman primates. Precise identification and definition of pericytes and pericyte subsets in humans may help us to better understand pericyte biology and develop new therapeutic approaches specifically targeting disease-associated pericyte subsets.

Astrocyte-Derived TGFβ1 Facilitates Blood-Brain Barrier Function via Non-Canonical Hedgehog Signaling in Brain Microvascular Endothelial Cells.

The blood–brain barrier is a specialized structure in mammals, separating the brain from the bloodstream and maintaining the homeostasis of the central nervous system. The barrier is composed of various types of cells, and the communication between these cells is critical to blood–brain barrier (BBB) function. Here, we demonstrate the astrocyte-derived TGFβ1-mediated intercellular communication between astrocytes and brain microvascular endothelial cells (BMECs). By using an in vitro co-culture model, we observed that the astrocyte-derived TGFβ1 enhanced the tight junction protein ZO-1 expression in BMECs and the endothelial barrier function via a non-canonical hedgehog signaling. Gli2, the core transcriptional factor of the hedgehog pathway, was demonstrated to modulate ZO-1 expression directly. By the dual-luciferase reporter system and chromatin immunoprecipitation, we further identified the exact sites on Smad2/3 that bound to the gli2 promotor and on Gli2 that bound to the zo-1 promotor. Our work highlighted the TGFβ1-mediated intercellular communication of astrocytes with BMECs in BBB, which shall extend current knowledge on the BBB homeostasis physiologically, and more importantly suggests TGFβ1 as a potential effector for future prevention and amelioration of BBB dysfunction.

Hyperglycemia modulates redox, inflammatory and vasoactive markers through specific signaling pathways in cerebral endothelial cells: Insights on insulin protective action

Type 2 diabetes is associated with major vascular dysfunctions, leading to clinical complications such as stroke. It is also known that hyperglycemia dysregulates blood-brain barrier homeostasis by altering cerebral endothelial cell function. Oxidative stress may play a critical role. The aim of this study was to evaluate the effect of hyperglycemia and insulin on the production of redox, inflammatory and vasoactive markers by cerebral endothelial cells. Murine bEnd.3 cerebral endothelial cells were exposed to hyperglycemia in the presence or not of insulin. Results show that hyperglycemia altered the expression of genes encoding the ROS-producing enzyme Nox4, antioxidant enzymes Cu/ZnSOD, catalase and HO-1 as well as Cu/ZnSOD, MnSOD and catalase enzymatic activities, leading to a time-dependent modulation of ROS levels. Cell preconditioning with inhibitors targeting PI3K, JNK, ERK, p38 MAPK or NFĸB signaling molecules partly blocked hyperglycemia-induced oxidative stress. Conversely, AMPK inhibitor exacerbated ROS production, suggesting a protective role of AMPK on the antioxidant defense system. Hyperglycemia also modulated both gene expression and nuclear translocation of the redox-sensitive transcription factor Nrf2. Moreover, hyperglycemia caused a pro-inflammatory response by activating NFĸB-AP-1 pathway and IL-6 secretion. Hyperglycemia reduced eNOS gene expression and NO levels, while increasing ET-1 gene expression. Importantly, insulin counteracted all the deleterious effects of hyperglycemia. Collectively, these results demonstrate that hyperglycemia dysregulated redox, inflammatory and vasoactive markers in cerebral endothelial cells. Insulin exerted a protective action against hyperglycemia effects. Thus, it will be of high interest to evaluate the benefits of antioxidant and anti-inflammatory strategies against hyperglycemia-mediated vascular complications in type 2 diabetes.

Peptide-Specific Engagement of Cerebrovascular Endothelial Cells Promotes Dysfunctional Calcium Signaling During Experimental Cerebral Malaria

Abstract Cerebral malaria is a potentially fatal complication associated with Plasmodium falciparum infection. Disease pathology includes blood brain barrier (BBB) breakdown, microvascular hemorrhage, edema and brainstem herniation. Studies in rodents (referred to as experimental cerebral malaria) have revealed that parasite-specific CD8+ T cells induce this pathology via engagement of cerebrovascular endothelial cells (ECs). In this study, we sought novel insights into the mechanisms underlying alterations in BBB structure and function during the development of ECM. We developed a novel transgenic reporter system that allowed us to simultaneously monitor calcium signaling in cerebrovascular ECs and parasite-specific CD8+ T cells. We then used intravital two-photon microscopy (TPM) to monitor BBB dynamics in real-time at the peak of disease. Within venous networks, we observed a significant increase in the magnitude and propagation of EC calcium signaling. Calcium wave propagation along lengthy stretches of cerebrovascular endothelium was associated with regions of parasite-specific CD8+ T cell engagement (both stable and dynamic). Recognition of cognate peptide-MHC on luminal and abluminal EC surfaces resulted in calcium fluxes within parasite-specific CD8+ T cells, which was followed temporally by calcium wave propagation in the associated vasculature. Importantly, this pathophysiological response was completely eliminated by depletion of T cells. We postulate that T cell-mediated induction of calcium waves along cerebral blood vessels promotes noncytopathic disruption of the BBB. Therapeutic manipulation of this activity might help restore BBB homeostasis and prevent neurological complications during ECM.

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies. Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy. Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease.

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies. Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy. Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

Disruption of Bmal1 Impairs Blood-Brain Barrier Integrity via Pericyte Dysfunction.

Circadian rhythm disturbances are well established in neurological diseases. However, how these disruptions cause homeostatic imbalances remains poorly understood. Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1) is a major circadian clock transcriptional activator, and Bmal1 deficiency in male Bmal1nestin-/- mice induced marked astroglial activation without affecting the number of astrocytes in the brain and spinal cord. Bmal1 deletion caused blood-brain barrier (BBB) hyperpermeability with an age-dependent loss of pericyte coverage of blood vessels in the brain. Using Nestin-green fluorescent protein (GFP) transgenic mice, we determined that pericytes are Nestin-GFP+ in the adult brain. Bmal1 deletion caused Nestin-GFP+ pericyte dysfunction, including the downregulation of platelet-derived growth factor receptor β (PDGFRβ), a protein necessary for maintaining BBB integrity. Knockdown of Bmal1 downregulated PDGFRβ transcription in the brain pericyte cell line. Thus, the circadian clock component Bmal1 maintains BBB integrity via regulating pericytes.SIGNIFICANCE STATEMENT Circadian rhythm disturbances may play a role in neurodegenerative disorders, such as Alzheimer's disease. Our results revealed that one of the circadian clock components maintains the integrity of the blood-brain barrier (BBB) by regulating vascular-embedded pericytes. These cells were recently identified as a vital component for the control of BBB permeability and cerebral blood flow. Our present study demonstrates the involvement of circadian clock component Bmal1 in BBB homeostasis and highlights the role of Bmal1 dysfunction in multiple neurological diseases.

A subset of cerebrovascular pericytes originates from mature macrophages in the very early phase of vascular development in CNS

Pericytes are believed to originate from either mesenchymal or neural crest cells. It has recently been reported that pericytes play important roles in the central nervous system (CNS) by regulating blood-brain barrier homeostasis and blood flow at the capillary level. However, the origin of CNS microvascular pericytes and the mechanism of their recruitment remain unknown. Here, we show a new source of cerebrovascular pericytes during neurogenesis. In the CNS of embryonic day 10.5 mouse embryos, CD31+F4/80+ hematopoietic lineage cells were observed in the avascular region around the dorsal midline of the developing midbrain. These cells expressed additional macrophage markers such as CD206 and CD11b. Moreover, the CD31+F4/80+ cells phagocytosed apoptotic cells as functionally matured macrophages, adhered to the newly formed subventricular vascular plexus, and then divided into daughter cells. Eventually, these CD31+F4/80+ cells transdifferentiated into NG2/PDGFRβ/desmin-expressing cerebrovascular pericytes, enwrapping and associating with vascular endothelial cells. These data indicate that a subset of cerebrovascular pericytes derive from mature macrophages in the very early phase of CNS vascular development, which in turn are recruited from sites of embryonic hematopoiesis such as the yolk sac by way of blood flow.

Dual role of ALCAM in neuroinflammation and blood–brain barrier homeostasis

Activated leukocyte cell adhesion molecule (ALCAM) is a cell adhesion molecule found on blood-brain barrier endothelial cells (BBB-ECs) that was previously shown to be involved in leukocyte transmigration across the endothelium. In the present study, we found that ALCAM knockout (KO) mice developed a more severe myelin oligodendrocyte glycoprotein (MOG)35-55-induced experimental autoimmune encephalomyelitis (EAE). The exacerbated disease was associated with a significant increase in the number of CNS-infiltrating proinflammatory leukocytes compared with WT controls. Passive EAE transfer experiments suggested that the pathophysiology observed in active EAE was linked to the absence of ALCAM on BBB-ECs. In addition, phenotypic characterization of unimmunized ALCAM KO mice revealed a reduced expression of BBB junctional proteins. Further in vivo, in vitro, and molecular analysis confirmed that ALCAM is associated with tight junction molecule assembly at the BBB, explaining the increased permeability of CNS blood vessels in ALCAM KO animals. Collectively, our data point to a biologically important function of ALCAM in maintaining BBB integrity.